Method for making nucleic acid probes

ABSTRACT

Modified nucleotides are provided which have the structure ##STR1## wherein R 1  is a reactive group derivatizabale with a detectable label, R 2  is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof. R 3  is hydrogen, methyl, bromine, fluorine or iodine, R 4  is hydrogen, an acid-sensitive, base-stable blocking group or an acyl capping group, R 5  is hydrogen or a phosphorus derivative, R 6  is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive. Methods of synthesizing the derivatizable nucleotide are disclosed, as are labeled polynucleotide probes prepared therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 945,876 filedDec. 23, 1986 which is a continuation-in-part of U.S. Ser. No. 807,624filed Dec. 11, 1985.

FIELD OF THE INVENTION

This invention relates generally to polynucleotide probes, and inparticular relates to a polynucleotide probe containing at least onelabeled, modified nucleotide.

DESCRIPTION OF RELEVANT LITERATURE

Meinkoth and Wahl, Anal. Biochem., (1984) 138:267-284, provide a reviewarticle of hybridization techniques. See also Leary et al., Proc. Natl.Acad. Sci. USA (1983) 80:4045-4049, for a description of the dot blotassay. Sandwich hybridization is described by Ranki et al., Curr. Top.Microbiol. Immunology (1983) pp. 308ff. See also Ranki et al., Gene(1983) 21:77-85, Virtanen et al., Lancet (1983) 381-383, and U.S. Pat.No. 4,486,539. European Pat. No. 123,300 describes biotin-avidincomplexes for use in detecting nucleic acid sequences. Sung, in Nucl.Acids Res. 9(22):6139-6151 (1981) and in J. Org. Chem. 47:3623-3628(1982), discusses the synthesis of a modified nucleotide and applicationof the modified structure in oligonucleotide synthesis. Modifiednucleotides are also discussed in Draper, Nucleic Acids Res.12:2:989-1002 (1984), wherein it is suggested that cytidine residues inRNA be modified so as to bind to reporter molecules. Later work suggestssimilar modification of cytidine residues in DNA (Anal. Biochem.157(2):199 (1986). European patent application Ser. No. 063879, filed 6Apr. 1982, and PCT application No. PCT/US84/00279 also describe modifiednucleotides and applications thereof.

BACKGROUND OF THE INVENTION

The increasing ease of cloning and synthesizing DNA sequences hasgreatly expanded opportunities for detecting particular nucleic acidsequences of interest. No longer must one rely on the use ofimmunocomplexes for the detection of pathogens, ligands, antigens, andthe like. Rather than detecting particular determinant sites, one candetect DNA sequences or RNA sequences associated with a particular cell.In this manner, diseases can be diagnosed, phenotypes and genotypes canbe analyzed, as can polymorphisms, relationships between cells, and thelike.

Analyses of DNA sequences typically involve the binding of an analytesequence to a solid support and hybridization of a complementarysequence to the bound sequence. The annealing and complexing stepsusually involve an extended period of time and require careful washingto minimize non-specific background signals. Applicants' co-pendingapplication Ser. No. 807,624, describes new techniques for analyzingnucleic acid sequences which are faster, minimize the number ofmanipulative steps, and provide for an increased signal to noise ratio.This application, a continuation-in-part of Ser. No. 807,624, thedisclosure of which is incorporated by reference herein, is directed inparticular to novel polynucleotide probes useful, inter alia, in thetechniques described in applicants' co-pending parent application.

The majority of polynucleotide probes in current use are radioactivelylabeled, e.g. with isotopes of hydrogen (³ H), phosphorus (³² P), carbon(¹⁴ C) or iodine (¹²⁵ I). These materials are relatively simple tosynthesize by direct inclusion of the radioactive moieties, e.g. bykinasing with ³² P-labeled ATP, equilibrating with tritiated water, orthe like. As is well known, however, use of such radioactive labels hasdrawbacks, and other detectable species which are not radioactive arepreferred.

In order to incorporate other, non-radioactive types of detectablespecies in a nucleotide, some sort of chemical modification of thenucleotide is required. It is widely recognized that nucleotidemodification is a difficult and sensitive procedure, as any modificationreaction has to be mild enough to leave the RNA or DNA molecules intact,while giving a modified nucleotide product which can participate innormal base pairing and stacking interactions. These considerationstypically limit nucleotide substitution positions to the 5-position of apyrimidine and the 8-position of a purine, as noted in the literature(see, e.g., European patent application Ser. No. 063879, cited supra).

Other considerations must also be taken into account. Base pairing maybe hindered during hybridization if the detectable label is at one endof the nucleotide chain rather than present at some point within it.Further, it has proved difficult to provide even non-radioactivelylabeled probes which may be inexpensively synthesized in large quantity.Thus, many known probes are limited in their potential applications.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to overcome theaforementioned disadvantages of the prior art.

It is another object of the present invention to provide a novelmodified nucleotide useful in the synthesis of labeled polynucleotideprobes.

It is still another object of the present invention to provide anucleotide modified at the 4-position of a pyrimidine base so as toinclude an alkylamine or other reactive moiety which is derivatizablewith a detectable label.

It is yet another object of the present invention to provide anucleotide so modified at the 4-position which is further modified atthe 5-position.

It is a further object of the present invention to provide a labeledpolynucleotide probe, at least one pyrimidine nucleotide of which ismodified at the 4-position so as to be derivatizable with a detectablelabel.

It is still a further object of the invention to provide methods ofmaking derivatizable alkylamine nucleotides.

It is another object of the invention to provide a method of using aprobe labeled according to the method of the present invention to detectthe presence of known nucleic acid sequences in a sample.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art of examination of thefollowing, or may be learned by practice of the invention.

In one aspect of the invention, a modified, derivatizable nucleotide isprovided having the structure of Formula 1: ##STR2## wherein R¹ is areactive group derivatizable with a detectable label, which reactivegroup may be amine, carboxyl or thiol and further may be protected forvarious synthetic manipulations, R² is an optional linking moiety suchas those typically used to label proteins, and includes an amide,thioether or disulfide linkage or a combination thereof, R³ is selectedfrom the group consisting of hydrogen, methyl, bromine, fluorine andiodine, R⁴ is hydrogen, an anchoring group which covalently binds thestructure to a solid support, or a blocking group such asdimethoxytrityl or pixyl, which blocking group is generally base-stableand acid-sensitive, R⁵ is hydrogen, an anchoring group which covalentlybinds the structure to a solid support, or a phosphorus derivativeenabling addition of nucleotides at the 3' position, and may be, forexample, PO₃ H₂, a phosphotriester, a phosphodiester, a phosphite, aphosphoramidite, H-phosphonate or a phosphorothioate, and R⁶ is H, OH,or OR where R is a functional group useful as a protecting moiety in RNAsynthesis, and x is an integer in the range of 1 and 8 inclusive.

In another aspect of the invention, a method of making the abovemodified nucleotide is provided including the step of derivatizing theR¹ moiety with a detectable label.

In still another aspect, a polynucleotide probes is provided using oneor more of the above modified nucleotides. The probe can be used toscreen a sample containing a plurality of single-stranded ordouble-stranded polynucleotide chains, and will label the desiredsequence, if present, by hybridization.

DETAILED DESCRIPTION OF THE INVENTION

"Derivatizable" nucleotides are nucleotides modified so as to include atthe 4-position of a pyrimidine a functional group which can react with adetectable label. An example of a derivatizable nucleotide is one whichhas been modified at the 4-position with an alkylamine moiety so that afree amine group is present on the structure.

"Derivatized" nucleotides are nucleotides in which the derivatizablefunctional group at the 4-position of the pyrimidine is bound,covalently or otherwise, directly or indirectly, to a detectable label.

"Alkylamine nucleotides" are nucleotides having an alkylamine group atthe 4-position of a pyrimidine, bound to the stucture in such a way asto provide a free amine group at that position.

A "polynucleotide" is a nucleotide chain structure containing at leasttwo nucleotides. The "polynucleotide probe" provided herein is anucleotide chain structure, as above, containing at least twonucleotides, at least one of which includes a modified nucleotide whichhas substantially the same structure as that given by Formula 1.

"Detectable label" refers to a moiety which accounts for thedetectablility of a complex or reagent. In general, the most commontypes of labels are fluorophores, chromophores, radioactive isotopes,and enzymes.

"Fluorophore" refers to a substance or portion thereof which is capableof exhibiting fluorescence in the detectable range. Typically, thisfluorescence is in the visible region, and there are common techniquesfor its quantitation. Examples of fluorophores which are commonly usedinclude fluorescein (usually supplied as fluorescein isothiocyanate[FITC] or fluorescein amine), rhodamine, dansyl and umbelliferone.

The nucleotide numbering scheme used herein is illustrated by Formulae2-5. ##STR3##

In a preferred embodiment, the substituents of the modified nucleotideof Formula 1 are as follows.

R¹, which is a reactive group derivatizable with a detectable label, ispreferbly --NH₂, --COOH or --SH.

R² is an optional linker moiety which contains an amide, thioether ordisulfide linkage, or a combination thereof. R² is preferably aheterobifunctional linker such as those typically used to bind proteinsto labels. In most cases, a free amino group on a protein or otherstructure will react with a carboxylic acid or activated ester moiety ofthe unbound R² compound so as to bind the linker via an amide linkage.Other methods of binding the linker to the nucleotide are also possible.Examples of particularly preferred linkers include. ##STR4## wherein xis an integer in the range of 1 and 8 inclusive.

As many be seen in Formula 1, the linker, if present, is attached to thenucleotide structure through an alkylamine functionality --NH--(CH₂)_(x)-- wherein x is an integer in the range of 1 and 8 inclusive, and thealkylamine functionality is present at the 4-position of the pyrimidinebase.

As noted above, R³ is hydrogen, methyl, bromine, fluorine or iodine.Thus, the base of the nucleotide is a pyrimidine optionally substitutedat the 5-position with the aforementioned R³ substituents.

R⁴ is typically hydrogen, if the modified nucleotide is a terminal 5'structure, or a suitable blocking group useful in polynucleotidesynthesis. Examples of suitable blocking groups include substituted andunsubstituted aralkyl compounds, where the aryl is, e.g., phenyl,naphthyl, furanyl, biphenyl and the like, and where the substituents arefrom 0 to 3, ususally 0 to 2, and include any non-interfering stablegroups, neutral or polar, electron-donating or withdrawing, generallybeing of 1 to 10, usually 1 to 6 atoms and generally of from 0 to 7carbon atoms, and may be an aliphatic, alicyclic, aromatic orheterocyclic group, generally aliphatically saturated, halohydrocarbon,e.g., trifluoromethyl, halo, thioether, oxyether, ester, amide, nitro,cyano, sulfone, amino, azo, etc.

In one or more steps during nucleotide chain synthesis, it may bedesirable to replace the hydrogen atom or blocking group at the R⁴position with a more stable, "capping" group. Suitable capping groupsinclude acyl groups which provide for stable esters. The acyl groups maybe organic or inorganic, including carboxyl, phosphoryl, pyrophosphoryl,and the like. Of particular interest are alkanoic acids, moreparticularly aryl-substituted alkanoic acids, where the acid is at least4 carbon atoms and not more than about 12 carbon atoms, usually not morethan about 10 carbon atoms, with the aryl, usually phenyl, substitutedalkanoic acids usually of from 8 to 12 carbon atoms. Various heteroatomsmay be present such as oxygen (oxy), halogen, nitrogen, e.g., cyano,etc. For the most part the carboxylic acid esters will be base labile,while mild acid stable, particularly at moderate temperatures belowabout 50° C., more particularly, below about 35° C. and at pHs greaterthan about 2, more particularly greater than about 4.

The modified nucleotide may also be attached to a support through the R⁴position so as to facilitate addition of labeled or unlabelednucleotides at the 3' (R⁵) position. In such a case, R⁴ is an anchoringgroup as will be described below. Covalent attachment to a support isalso preferred during sample screening, as the time and complexity ofseparating the hybridized nucleotide chains from the sample issubstantially reduced. When the modified nucleotide of Formula 1 isbound to one or more additional nucleotides at the 5' position, the R⁴substituent is replaced with such additional nucleotides which are boundthrough their 3' phosphate groups.

R⁵, as noted, is hydrogen or a phosphorus derivative such as PO₃ H₂, aphosphotriester, a phosphodiester, a phosphite, a phosphoramidite, anH-phosphonate or a phosphorothioate suitable for polynucleotidesynthesis, which derivative enables sequential addition of nucleotidesat the 3' position. More generally, such phosphorus derivatives aregiven by Formula 9 and Formula 10: ##STR5## wherein X is preferablyhydrogen or an aliphatic group, particularly a saturated aliphaticgroup, a β-heterosubstituted aliphatic group, where the β-substituent isan electron-withdrawing group which readily participates inβ-elimination, either as the leaving group or the proton-activatinggroup, substituted methylene, where the substituent may vary widely andsupports a negative charge on the methylene through inductive orresonating effects; aryl; and aralkyl. Depending on the nature of thephosphorus functionality, one group may be chosen over another. Thus,depending upon whether a phorphorchloridite, phosphoramidite, phosphate,thiophosphate, phosphite, or the like, is employed, particular phosphoroester groups will be preferred.

Similarly, the groups employed for Y will depend upon the nature of thephosphorus derivative employed for oligomerization. When thephosphoramidite is employed, Y will have the formula --NT¹ T², where T¹and T² may be the same or different and may be hydrocarbon or have from0 to 5, usually 0 to 4 heteroatoms, primarily oxygen as oxy, sulfur asthio, or nitrogen as amino, particular tert.-amino, NO₂ or cyano. Thetwo T's may be taken together to form a mono- or polyheterocyclic ringhaving a total of from 1 to 3, usually 1 to 2 heteroannular members andfrom 1 to 3 rings. Usually, the two T's will have a total of from 2 to20, more usually 2 to 16 carbon atoms, where the T's may be aliphatic(including alicyclic), particularly saturated aliphatic, monovalent, or,when taken together, divalent radicals, defining substituted orunsubstituted heterocyclic rings. The amines include a wide variety ofsaturated secondary amines such as dimethylamine, diethylamine,diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine,methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine,methylcyclohexylmethylamine, butylcyclohexylamine, morpholine,thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,piperazine and similar saturated monocyclic nitrogen heterocycles.

R⁵ may also represent a point of attachment for one or more additionalnucleotides at the 3' position. In that case R⁵ is phosphate, as suchadditional nucleotides are typically bound through a phosphate group.

As at the 5' position, the modified nucleotide may be attached to asupport through the 3' position, i.e. through R⁵. When the nucleotidethus attached to a support, R⁵ is an anchoring group as will bedescribed below.

R⁶, in the case of deoxyribose, is H; in the case of ribose, is OH;,and, during RNA synthesis, is a suitable blocking group which protectsthe --OH moiety from modification. Blocking groups useful here generallyinclude those given above for R⁴, and the specific choice of blockinggroup will be apparent to one skilled in the art. Examples of blockinggroups which are preferred at the R⁶ position during RNA synthesisinclude silyl ethers such as t-butyldimethylsilyl, substituted methylethers, o-nitrobenzyl ether, esters such as levulinic ester, and thefollowing pyranyl structures given by Formula 11 (tetrahydropyranyl) andFormula 12 (4-methoxytetrahydropyranyl): ##STR6##

A particularly preferred blocking group is ortho-nitrobenzyl. Additionalexamples of suitable blocking groups may be found in Green, T.W.,Protective Groups in Organic Synthesis, New York: Wiley & Sons, 1981.

The modified nucleotide will normally be derivatized with a label in amanner which will allow for detection of complex formation. A widevariety of labels may be used, and one or another label may be selecteddepending upon the desired sensitivity, the equipment available formeasuring, the particular protocols employed, ease of synthesis, and thelike. Labels which have found use include enzymes, fluorescers,chemiluminescers, radionuclides, enzyme substrates, cofactors or suicideinhibitors, specific binding pair members, particularly haptens, or thelike. The molecule involved with detection may be covalently bound tothe modified nucleotide or indirectly bound through the intermediacy ofa specific binding pair, i.e. ligand and receptor. Examples of ligandsand receptors include biotin-avidin, hapten-antibody, ligand-surfacemembrane receptor, metal-chelate, etc.

As suggested above, it is preferred that the modified nucleotide becovalently bound to a support of either the R⁴ or R⁵ positions foroligonucleotide synthesis. A wide variety of supports may be used,including silica, Porasil C, polystyrene, controlled pore glass (CPG),kieselguhr, poly(dimethylacrylamide), poly(acrylmorpholide), polystyrenegrafted onto poly(tetrafluoroethylene), cellulose, Sephadex LH-20,Fractosil 500, etc.

Depending on the nature of the support, different functionalities willserve as anchors. As noted above, these "anchoring" groups are at eitherthe 3' or the 5' position, i.e. at either the R⁵ or R⁴ positions,respectively. For silicon-containing supports, such as silica and glass,substituted alkyl or aryl silyl compounds will be employed to form asiloxane or siloximine linkage. With organic polymers, ethers, esters,amines, amides, sulfides, sulfones and phosphates may find use. For arylgroups, such as polystyrene, halomethylation can be used forfunctionalization, where the halo group may then be substituted by oxy,thio (which may be oxidized to sulfone), amino, phospho (as phosphine,phosphite or phosphate), silyl or the like. With a diatomaceous earthelement (e.g., kieselguhr), activation may be effected by a polyacrylicacid derivative and the active functionality reacted with amino groupsto form amine bonds. Polysaccharides may be functionalized withinorganic esters, e.g. phosphate, where the other oxygen serves to linkthe chain. With polyacrylic acid derivatives, the carboxyl or side chainfunctionality, e,g., N-hydroxyethyl acrylamide, may be used inconventional ways for joining the linking group.

The modified nucleotide of Formula 1, as previously suggested, can beused as a substrate for synthesis of polynucleotide probes. Additionalnucleotides may be sequentially added at the 5' position by, forexample, the phosphoramidite method of Beaucage and Caruthers,Tetrahedron Lett. 22(20):1859-62 (1981) or the phosphotriester method ofItakura, J. Biol. Chem. 250:4592 (1975), or the like, or at the 3'position by the method of Belagaje and Brush, Nuc. Acids Research10:6295 (1982), or both. The nucleotrides which are sequentially addedmay be unlabeled, or they may be modified according to Formula 1 andderivatized with a label at the R¹ moiety. Accordingly, one or morelabels may be present within a polynucleotide chain rather than at oneend.

This polynucleotide probe includes at least one modified nucleotidehaving substantially the same structure as that given by Formula 1, i.e.including at least one modified nucleotide having the structure given byFormula 13: ##STR7## wherein R¹ is a reactive group derivatized with adetectable label, R² is an optional linking moiety including an amide,thioether or disulfide linkage or a combination thereof, R³ is selectedfrom the group consisting of hydrogen, methyl, bromine, fluorine andiodine, R⁶ is H, OH, or OR where R is an acid-sensitive, base-stableprotecting group and x is an integer in the range of 1 and 8 inclusive.The polynucleotide probe may have a single label of a plurality oflabels, depending upon the nature of the label and the mechanism fordetection. Where the label is fluorescent, for example, a distance of atleast 3 to 12 Angstroms should be maintained between fluorescent speciesto avoid any fluorescence quenching.

Such labeled polynucleotide probes may be used in the assays describedin applicants' co-pending application Ser. No. 807,624, or in any numberof other applications, includiing conjugation with enzymes, antibodiesand solid supports. An example of one such use of applicants' noveloligonucleotide probes is in the detection of a known sequence of DNA.The probe may be prepared so as to be attached, for example, to astandard latex solid support or to an avidin support in the case ofbiotin-labeled probes. Sample containing single-stranded ordouble-stranded DNA sequences to be analyzed is caused to contact theprobe for a time sufficient for hybridized nucleic acid complexes toform, and any such complexes are detected by means of the fluorescent,biotin or otherwise detectable label.

Synthesis of the modified nucleotide: The present invention also relatesto a method of synthesizing the novel modified nucleotide of Formula 1.In the preferred embodiment, a pyrimidine nucleotide is provided whichhas the structure of Formula 14 or Formula 15: ##STR8## wherein R³ is asgiven above, R⁴ and R⁵ are hydrogen, and R⁶ is OH or H. The 5' positionof the sugar ring--and the 2' position as well if the sugar is riboserather than deoxyribose--is then protected against modification duringsubsequent reaction steps by addition of a dimethoxytrityl group (seeExample 3) or other suitable protecting group, the addition reactionallowed to proceed for a time sufficient to ensure substantialcompleteness. Similarly, the 3' hydroxyl group is protected with a silylor other suitable functionality (see Example 4).

Examples of particularly suitable protecting groups include those setforth above as "R⁶ ", i.e., substituted methyl ethers, esters, pyranylsand the like.

When the nucleoside is thymine or uracil, or uracil modified at the5-position by an R³ substituent, i.e. a pyrimidine or substitutedpyrimidine which has an oxy rather than an amino substituent at the4-position, the carbonyl is converted to an amine moiety by, forexample, reaction with an activating agent such as1-(mesitylene-2-sulfonyl)-tetrazole (MS-tet) or other suitablecondensing reagent. Activating agents for use herein also include othersulfonyl compounds given by the formula E₁ --SO₂ --E₂ wherein E₁ istetrazoyl, nitrotriazoyl, triazoyl, imidazoyl, nitroimidazoyl, or thelike, and E₂ is an aryl or substituted aryl group such as mesitylene,etc. Another class of suitable activating agents is given by Formula 16:##STR9## wherein E₁ is as defined above. In Formula 16b. E₁ is presentin a solution containing the activating agent but is not bound thereto,and X is a halogen substituent, preferably chlorine. In general, anyactivating agent may be used and may include one or more halogensubstituents, preferably chlorine, on the ring structure which afterreaction can be displaced by ethylene diamine or like reagent. Thisconversion is followed by reaction with an alkyldiamine such asethylenediamine to give a nucleotide having a --NH--(CH₂)_(x) NH₂functionality at the 4-position of the pyrimidine ring (see Examples 5,6). The free amine group so provided is then optionally reacted withcaproic acid, an actiivated caproic acid ester, or with a caproic acidderivative such as 6-aminocaproic acid, in order to ensure sufficientspacing between the nucleotide and the detectable label to be attachedat the R¹ moiety. The caproic acid or related compound may be labeledprior to attachment (see Example 7) or subsequently.

When the nucleoside is cytosine or a 5-modified cytosine, i.e.substituted with an R³ other than hydrogen, the exocyclic aminofunctionality can be converted to an N⁴ -aminoalkyl or N⁴ -aminoarylcytosine by reaction with an aryl sulfonyl chloride followed by reactionwith an alkyl- or aryldiamine (Scheme I). See, e.g., Markiewicz, W. T.and R. Kierzek, 7th Intl. Round Table, pp. 32 and 72 (1986).Alternatively, preparation of N⁴ -substituted cytosine may be effectedusing a bisulfite-catalyzed exchange reaction. See Schulman, L. H. etal., Nuc. Acids Res. 9:1203-1217 (1981) and Draper, D. E., Nuc. Acids.Res. 12:989-1002 (1984). ##STR10##

Alternatively, where the alkylamine group is more than about 6 carbonatoms long, the free amine group thereof may directly bond to a suitabledetectable label.

The synthesis may further include removal of the dimethoxytrityl orother protecting groups with acid, followed by, if desired,phosphorylation or phosphitylation of the 3' position in preparation forsequential addition of nucleotides.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples which follow are intendedto illustrate and not limit the scope of the invention, which is definedby the scope of the appended claims. Other aspects, advantages andmodifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains.

EXAMPLES EXAMPLE 1 Labeling of Caproic Acid Derivative ##STR11##

To 1 mmole of fluorescein isothiocyanate in 5 ml of DMF was added 2mmole of 6-aminocaproic acid and 540 μl of triethylamine. After 24 h atroom temperature, the product was isolated by preparative thin layerchromatography (Warner and Legg, Inorg. Chem. 18:1839 (1979)). The driedproduct was suspended in 10 ml of 1:1 DMF/THF (v/v) to which 1.5 mmoleof N-hydroxy succinimide and 1 mmole of dicyclohexylcarbodiimide wereadded. After 18 h at room temperature the solution was filtered throughglass wool and diluted to a 0.2M final concentration of A with DMF(assuming a 100% yield from step 1).

EXAMPLE 2 6-N⁴ -(2-Aminoethyl)-Deoxycytidine ##STR12##

An alkylated derivative of deoxycytidine, N⁴ -(2-aminoethyl)deoxycytidine (B) was prepared from properly protected deoxyuridine viathe 4-tetrazoyl derivative as described by Reese and Ubasawa,Tetrahedron Lett. 21:2265 (1984). This latter derivative was convertedto B by displacement of the tetrazoyl moiety with ethylene diamineessentially as described by Sung, J. Org. Chem. 47:3623 (1982) andMaggio et al., Tetrahedron Lett. 25:3195 (1984). The corresponding5'-DMT-3'-phosphoramidite N⁴ -(2-N-trifluoroacetylaminoethyl)deoxycytidine was prepared by blocking the alkylamine withtrifluoroacetic anhydride and then preparing the correspondingN,N-diisopropyl phosphoramidite as described (Beaucage and Caruthers,supra; McBride and Caruthers, Tetrahedron Lett. 24:245 (1983)).

EXAMPLE 3 5'-Dimethoxytrityl-2'-Deoxyuridine ##STR13##

To 2-Deoxyuridine (10 g, 44 mmole) dried by coevaporation of pyridineand suspended in pyridine (100 ml) was added 18.4 g (54 mmole)4,4'-dimethoxytrityl chloride (DMT-Cl). The reaction was allowed toproceed for 18 h at room temperature, and 100 ml methanol was added todeactivate excess DMT-Cl. Most of the pyridine was then removed invacuo, and the residue, dissolved in 500 ml ethyl acetate, was washedwith saturated aqueous NaHCO₃ (3×500 ml). The organic phase was driedover solid Na₂ SO₄ and evaporated to dryness. The residue was purifiedby flash chromatography on silica gel to give 18.0 g (77%) of5'-dimethoxytrityl-2'-deoxyuridine (C).

EXAMPLE 45'-O-(4,4'-Dimethoxytrityl)-3'-t-Butyldimethylsilyl-2'-Deoxyuridine##STR14##

To 18 g (34 mmole) of C in 200 ml DMF was added imidazole (5.8 g, 85mmole) with rapid stirring to assure complete dissolution.t-Butyldimethylsilyl chloride (7.65 g, 51 mmole) dissolved in a smallvolume of DMF was added dropwise with stirring and the reaction wasallowed to proceed in the dark for 18 h at room temperature. Thereaction mixture was diluted with ethyl acetate (250 ml) and extractedwith NaHCO₃ (3×250 ml). The organic phase was dried over Na₂ SO₄ andevaporated to dryness. The residue was purified by flash chromatographyon silica gel to give 15.0 g (68% yield) of5'-O-(4,4'-dimethoxytrityl-3'-t-butyldimethylsilyl-2'-deoxyuridine (D).

EXAMPLE 54-(1,2,3,4-Tetrazol-1-yl)-[5'-(4,4'-Dimethoxytrityl)-3'-t-Butyldimethylsilyl-β-D-2'-Deoxyribosyl]Pyridine-2(1H)-one ##STR15##

To 15.0 g (23 mmole) of D, dried by coevaporation of pyridine anddissolved in pyridine (50 ml) was added diphenylphosphate (2.9 g, 11.5mmole) dissolved in pyridine (5 ml). 1-(Mesitylene-2-sulfonyl)-tetrazole(MS-tet) (15.5 g, 61.5 mmole) dissolved in pyridine (45 ml) was addedand the reaction mixture allowed to proceed in the dark for 18 h at roomtemperature. To the dark brown reaction mixture was added 25 ml water.After 30 min, the product was concentrated under reduced pressure. Theresidue was dissolved in 250 ml methylene chloride, washed with anaqueous NaHCO₃ solution (3×250 ml), dried over Na₂ SO₄, and the solventwas removed under reduced pressure in the presence of toluene. Theresidue was purified by flash chromatography on silica gel to give 10.0g (62%) of4-(1,2,3,4-Tetrazol-1-yl)-[5'-(4,4'-dimethoxy-trityl)-3'-t-butyldimethylsilyl-β-D-2'-deoxy-ribosyl]-pyridine-2(1H)-one(E).

EXAMPLE 64-N-(2-Aminoethyl)-5'-Dimethoxytrityl-3'-t-Butyldimethylsilyl-2'-Deoxycytidine##STR16##

To a solution of ethylene diamine (9.3 ml, 143 mmole) in dioxane (100ml) cooled to 5° C. was added E (10.0 g, 14.3 mmole) and left for onehour. The solvent was removed at reduced pressure and the residue wascoevaporated with toluene to remove excess ethylene diamine. The productwas purified by chromatography on a silica gel column, eluted with12-20% methanol in methylene chloride to give 7.15 g (75%) of4-N-(2-aminoethyl)-5'-dimethoxytrityl-3'-t-butyldimethylsilyl-2'-deoxycytidine(F). The product was shown to react positively with ninhydrin,confirming the presence of a free amine moiety.

EXAMPLE 7 N⁴-(N-FMOC-6-Aminocaproyl-2-Aminoethyl)-5'-Dimethyltrityl-3'-t-Butyldimethylsilyl-2'-Deoxycytidine##STR17##

To a solution of F (6.5 g, 9.6 mmole) in pyridine (50 ml) was addedN-FMOC-6-aminocaproic acid (4.26 g, 12 mmole) (FMOC represented bystructure H) and DCC (2.96 g, 14.4 mmole). After 3 h, the reaction wascomplete as judged by tlc (silica in 10% methanol/methylene chloride).Pyridine was removed at reduced pressure. The residue was extracted withethyl acetate, insoluble dicyclohexylurea (DCHU) filtered off and thesolvent removed. The product was isolated by silica gel chromatographyeluted with 4% methanol in methylene chloride affording 7.3 g (70%) ofN⁴-(N-FMOC-6-amino-caproyl-2-amino-ethyl)-5'-dimethyltrityl-3'-t-butyldi-methylsilyl-2'-deoxycytidine(G).

EXAMPLE 8 ##STR18##

A solution of tetrabutylammonium fluoride (15 mmole, 15 ml of a 1Msolution in THF) and aqueous HF (1.05 ml of a 50% aqueous solution) weremixed and dried by coevaporation of pyridine. The residue was dissolvedin pyridine (15 ml) and added to G (7.2 g, 7.3 mmole) which wasdissolved by sonication. After 18 hours at 4° C. the reaction mixturewas diluted with 200 ml methylene chloride. Concentrated aqueous NaHCO₃was carefully added followed by solid NaHCO₃, added gradually so as toneutralize the HF/pyridine. After drying over Na₂ SO₄, the organic phasewas concentrated to an oil, which was subjected to silica gelchromatography. The product N⁴-(N-FMOC-6-aminocaproyl-2-aminoethyl)-5'-dimethoxytri-tyl-2'-deoxycytidine(I) was eluted with 5-6% methanol in methylene chloride to give an 86%yield (6.0 g).

EXAMPLE 9 ##STR19##

To 5.1 g (5.7 mmole) of I in methylene chloride containing(diisopropylethylamine) was added ##STR20##(chloro-N,N-diisopropylaminomethoxy phosphine, 1.3 ml [1.2 eq.], K) at0° C. under argon. After 1 hr, ethyl acetate (200 ml) was added andwashed with 80% saturated aqueous sodium chloride; after drying of theorganic phase over Na₂ SO₄, the product in methylene chloride was addeddropwise to hexane at -40° C. to precipitate 4.43 g (75%) of J.

EXAMPLE 10 Probe Preparation (Fluorescein Label)

Synthetic oligonucleotides were prepared by an automated phosphoramiditemethod as described in Warner et al., DNA 3:401 (1984). Purification wascarried out according to Sanchez-Pescador and Urdea, DNA 3:339 (1984).

The aminoethyl derivative of deoxycytidine as prepared in Example 2 wasincorporated by standard coupling procedures during the oligonucleotidesynthesis and the purified modified oligonucleotides were used forincorporation of a fluorescein label as follows. To a dried sample (3-5OD 260 units) of the aminoethyl deoxycytidine containing oligomer wereadded 50 μl of DMF and 25 μl of the 0.2M stock solution of A describedabove. After 18 h at room temperature, the solution was partiallypurified by Sephadex G-10 chromatography eluted with water, dried andfurther purified by polyacrylamide gel, as above.

EXAMPLE 11 Probe Preparation (Biotin Label)

Using the probes containing aminoethylcytidine as prepared in theprevious example, biotin labeling was achieved as follows. Theoligonucleotide (3-5 OD 260 units) was taken up in 50 μl 0.1M sodiumphosphate, pH 7.0 and 50 μl of DMF to which 100 μl of a DMF solutioncontaining 1 mg of a "long chain" N-hydroxysuccinimidyl biotin (PierceChemical) was added. After 18 h at room temperature, the biotinylatedprobe was purified as described for the fluorescein labeled probe.

EXAMPLE 12 Synthesis of Horseradish Peroxidase (HRP): DNA Conjugates

Sequence 1 (5'-[LCA]CTGAACGTTCAACCAGTTCA-3') where LCA=N⁴(6-aminocaproyl-2-aminoethyl)-deoxy cytidine) was synthesized chemicallyand purified as described elsewhere (Warner, et al. (1984) DNA 3, 401).To 10 OD 260 units dissolved in 50 μl of water were added 10 μl of 1.0 Msodium borate, pH 9.3, and 500 μl of distilled dimethylformamidecontaining 20 mg of p-phenylene diisothiocyanate. The solution wasvortexed and set for 2 hr at room temperature in the dark. Approximately3 ml of n-butanol was then added. After vortexing, adding 3 ml of water,and vortexing again, the tube was centrifuged and the yellowish upperlayer discarded. The extraction process was repeated with subsequentn-butanol additions until an final volume of approximately 50 μl wasobtained. The butanol was removed by evacuation, then 10 mg of HRP in200 μl of 0.1 M burate, pH 9.3, was added. The mixture was vortexed,then set at room temperature overnight in the dark.

Separation of the HRP-DNA conjugate from free enzyme and DNA wasachieved on a 7% polyacrylamide gel. The 250 μl reaction mixture wasquenched with 100 μl of 25% glycerol, 0.5% SDS, 0.5% bromophenol blue,2.5 mM EDTA. The solution was then distributed into 10 lanes of a 20×200.15 cm gel and run at 60 mAmps under standard conditions (Maxam, A.,and Gilbert, W., (1980) Methods in Enzymol 65, 499-560) until thebromophenol blue was about 2/3 down the gel. The gels were set on BakerF-254 silica 60 plates that had been covered with Saran Wrap (Dow) andexamined with a handheld UV-short wavelength lamp held above. Picturesof the UV-shadowed bands were taken with a Polaroid MP-4 camera systemfitted with a Kodak No. 59 green filter, after which the bands were cutout with a razor blade. The bands were put into a 10-ml Bio-Radpolypropylene econo-columns to which 3 ml of 0.1 M sodium phosphate, pH7.5, was added, then set at room temperature overnight.

The contents of the column were filtered through the frit at the columnbottom into an Amicon centricon microconcentrator that had been washedtwice with distilled water. The HRP-DNA conjugate was then concentratedby centrifugation at 3500 rpm and washed twice with 1× PBS also bycentrifugation. The final solution was then stored at 4° C.

EXAMPLE 13 Assay for HBV DNA Using HRP-DNA Probe and a BiotinylatedProbe Bound to an Avidin Bead

Biotin labeled probe (B'; 1000 pmoles in 66.7 μl of water) was combinedwith 5 ml of a 0.25% (w/v) solution of 0.8 μ avidin beads (Pandexlaboratories), 1 ml of 20× SSC, 0.5 ml of 1% NP40 and 0.6 of 1 mg/mlpolyA. After 1 h at 37° C., the beads were washed twice bycentrifugation with 4× SSC, 0.1% NP40 then stored in 2.5 ml of thissolution. The HBV analyte (described in copending application Ser. No.807,624, previously incorporated by reference) in 3 μl water was dilutedinto 10 μl of 4× SSC, 1% SDS, 0.5 M NaOH and 1.5 pmoles of the labelingand capturing probe sets. The mixture was heated to 95° C. for 10 min.,cooled on ice and neutralized with 5 μl of 1 M acetic acid, then 10 μlof the biotin probe beads were added and the solution was incubated at37° C. for 1 h.

The beads were washed twice by centrifugation with 4× SSC, 0.1% NP40,then taken up in 50 μl of 0.1% NP40, 1 mg/ml polyA, 10 mg/ml BSA, 1× PBScontaining 1 pmole of HRP-DNA conjugate and set a 37° C. for 1 h. Thebeads were washed with 0.1% NP40, 1× PBS three times then transferred in50 μl to a microtiter dish. To each well, 50 μl of fresh OPD solution(98 mg OPD (O-phenylenediamine), 20 μl of 30% H₂ O₂ in 10 ml of 50 mMsodium citrate pH 5.0) was added, mixed and set 5 min. at 37° C. Theabsorbances were recorded on a microtiter plate reader. Controlhybridizations contained no HBV analyte.

                  TABLE 4                                                         ______________________________________                                        Condition      Absorbance Reading                                             ______________________________________                                        1         pmole    >2                                                         0.1       pmole    >2                                                         0.01      pmole    0.88 ± 0.23                                             1         fmole    0.20 ± 0.05                                             0.1       fmole    0.07 ± 0.03                                             NO ANALYTE     0.01 ± 0.01                                                 ______________________________________                                    

We claim:
 1. A method of making a polynucleotide probe, comprising:(a)providing a modified nucleotide given by the structure ##STR21##wherein: R¹ is a protected or unprotected --NH₂, --COOH or --SH group;R² is a bond or an amide, thioether or disulfide linking moeity; R³ ishydrogen, methyl, bromine, fluorine or iodine; R⁴ is selected from thegroup consisting of hydrogen, acid-sensitive, base-stable blockinggroups and acyl capping groups; R⁵ is a phosphorus derivative selectedfrom the group consisting of PO₃ H₂, phosphotriesters, phosphodiesters,phosphites, phosphoramidites, H-phosphonates and phosphorothioates andmay be bound through R⁵ to a solid support; R⁶ is H, OH, or OR where Ris an acid-sensitive, base-stable protecting group; and x is an integerin the range of 1 and 8 inclusive; and (b) sequentially addingnucleotides to either the 3' and of said modified nucleotde, the 5' end,or both, wherein a fraction of the added nucleotides comprise aplurality of said modified nucleotides, thereby providing apolynucleotide probe having said modified nucleotides incorporated atpredetermined, spaced apart positions.
 2. The method of claim 1, furtherincluding, after (b):(c) labeling the modified nucleotides within saidpolynucleotide probe by derivatization with a labeling species at the R¹moiety.
 3. The method of claim 1, further including, prior to step (a),labeling each of said modified nucleotides to be incorporated into saidprobe by derivatization with a labeling species at the R¹ moiety.
 4. Themethod of claim 1, wherein R¹ is a protected or unprotected --NH₂ group.5. The method of claim 4, wherein R² is a bond or an amide linkingmoiety.
 6. The method of claim 5, wherein R⁴ and R⁶ are hydrogen.